cDNA integration into Plasmid

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Ultrashogun
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cDNA integration into Plasmid

Post by Ultrashogun » Wed Oct 18, 2006 5:25 pm

How is a fragment of cDNA integrated into a plasmid although it does not have complementary extremities due to originating from restrictive endonucleasic digestion?

oppox
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Post by oppox » Wed Oct 18, 2006 7:18 pm

Ive done a lab where we created a cDNA library I think but it was a long time ago and I dont remember the details so here is a link to something similar.

http://courses.washington.edu/dmandoli/ ... ibrary.pdf

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canalon
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Post by canalon » Wed Oct 18, 2006 9:36 pm

If you used a PCR step with Taq polymerase, TA cloning is an option. Blunt end cloning is always possible, although the results are not very good.
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Ultrashogun
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Post by Ultrashogun » Wed Oct 18, 2006 9:56 pm

oppox wrote:Ive done a lab where we created a cDNA library I think but it was a long time ago and I dont remember the details so here is a link to something similar.

http://courses.washington.edu/dmandoli/ ... ibrary.pdf


Thank you, that gave me the answer, which is:

add linkers to all the cDNAs. These linkers are short oligonucleotide pieces of
dsDNA that contain a restriction site within them. Add the enzyme DNA ligase,
which will ligate the linkers to the ends of the cDNAs.

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